Good methods to interrogate, and ideally perturb, neural systems at the network level have been elusive until recently. The advent of biochemical breakthroughs, namely neuronally-targeted genetically-encoded calcium reporters, such as GCaMP, and light-activated channels, such as channelrhodopsin, have enabled ?all-optical? electrophysiology where experiments can be conducted that involve simultaneous, or closely-sequenced, stimulation of multiple neurons and readout of an entire field of cortical or subcortical tissue. Intelligent Imaging Innovations, Inc (3i) has developed computer-generated holography, in conjunction with the laboratory of Dr Valentina Emiliani at Paris Descartes University, since 2011. The collaboration has been a success: 3i has since distributed over 25 holography systems to research laboratories in North America and Europe, which have already produced multiple publications, with more in preparation and review. Recently, 3i has also started working with the laboratories of Drs Emily Gibson and Diego Restrepo at the University of Colorado Anschutz Medical Campus to evaluate the feasibility of providing a commercial source for a novel two-photon fiber-coupled microscope that incorporates an electrowetting lens for remote focusing. Drs Gibson and Restrepo are advancing the fundamental photonics research, partially supported by the BRAIN U01 ?Controlled neuronal firing in vivo using two photon spatially shaped optogenetics.? Dr Emiliani (and her collaborator, Dr Serge Picaud), continue to advance computer-generated holography with support from the BRAIN U01 ?Three Dimensional Holography for Parallel Multi-target Optogenetic Circuit Manipulation? and other funding. Preliminary results, described in this application, suggest that 3i can couple its multiphoton holography through the fiberscope in addition to performing scanning multiphoton microscopy, allowing neuroscientists to stimulate individual neurons at multiple layers of cortex and monitor activity of a substantial field in freely-moving animal. 3i proposes to assess the feasibility of developing a compact, modular turn-key system for in vivo all-optical electrophysiology. The system would incorporate the necessary pulse compression, scanning pathway, detection pathway, computer-generated holography, and control software. We believe a commercial solution which incorporates the many collective years of expertise of 3i and its collaborators in the fields of multiphoton imaging, computer-generated holography, and fiber-coupled microscope design and assembly will enable many more laboratories, particularly those without extensive optical engineering resources, to advance brain understanding faster and fulfill one of the BRAIN Initiative directives, which is to provide wide dissemination of the technologies developed under the initiative. In order to provide such a solution, however, 3i must engage in further research and development to translate the benchtop results of Dr Gibson and Restrepo?s laboratories into a candidate for commercialization.